| Cytochrome c Oxidase (CcO) is the terminal enzyme of the redox respiratory chain in mitochondria and many aerobic bacteria. It catalyzes electron transfer from cytochrome c to molecular oxygen, reducing the latter to water. The enzyme is a complex metalloprotein embedded in the membrane and comprises several subunits and metal active sites. The CuA center in subunit II is essential to the physiological function of the enzyme because the electron from cytochrome c enters the CcO firstly via this site. Defects in cytochrome c oxidase can lead to many diseases, and several mutations in gene encoding the enzyme have been found in Alzheimers'disease and Cancers. In this work, we have expressed and isolated the native subunit II soluble CuA domain of CcO from Paracoccus versutus and its mutant proteins at sites Trp121 and Val162. Using spectral and kinetic techniques, we studied the effects of different mutation on structure, property and electron transfer function of the protein. The conformation transitions caused by mutation or other physical and chemical factors have also been reported. The followings are the main results.(I) Expression, purification and characterization of the soluble CuA domain of CcO The native soluble CuA domain of Paracoccus versutus has been expressed in E.coli BL21 (DE3). Almost the entire subunit II fragment was in inclusion bodies. After refolding in vitro, recombination of metal center and purification with Mono Q column running with a Pharmacia FPLC instrument, the soluble purple sample was obtained. The purified protein showed a single band on SDS-PAGE analysis, and the molecular mass of protein was measured accurately with electrospray mass spectrometry. The UV-visible and CD spectra were used to characterize the active site and secondary structure of the protein. The absorption peaks at 360, 480, 530 and 810 nm show a typical purple CuA center feature, and the far-UV CD spectra shows a negative peak at 215 nm, which presents a typical (-sheet structure. These characteristics are similar to that of other native and engineered CuA protein. The maximum of 342 nm in fluorescence emission spectrum of the protein shows that its fluorescence can be mainly assigned to the tryptophan residues. The efficient energy transfers were observed in the soluble CuA domain, including the energy transfer from Tyr to Trp residue and that from Trp to the CuA center.The activity of protein was demonstrated by monitoring the oxidation process of the reduced cytochrome c (Cyt c) by the soluble CuA domain.(II) The effects of mutation at Trp-121 on protein's structure and functionTrp121 is one member of the highly conserved aromatic cluster (Trp121, Tyr122, Trp123, Tyr125 and Tyr127) in CcO sdII. It locates at the surface of subunit II about 5 ? above the CuA center and interacts with several different CuA ligands. Functionally, the Trp121 has been considered as the electron entry site from Cyt c to CcO. However, the contribution of Trp121 to protein's structure and property had never been explored. The mechanism of transfer electron is also not clear.Using the site-directed technique of protein engineering, we have constructed and expressed the mutant proteins of W121Y, W121L and (W121 in soluble domain of subunit II of CcO from Paracoccus versutus. The W121Y mutant can refold properly and reform the CuA center almost completely. However, the (W121 and W121L mutant proteins lost the ability to reconstitute CuA center and mainly refold as another conformation. Their characteristics in CD spectrum are double negative peaks of 208 nm and 220 nm, which suggest the relative change in abundance of (-helices and (-sheets in those proteins. The conversion of CuA center to type II Cu2+ coordination is corroborated by EPR and Raman spectroscopy. Importantly, the mutations at Trp121 affect the electron transfer (ET) rate of the protein with Cyt c. The results from stopped-flow rapid kinetic studies show that the ET rate between the W121Y mutant and Cyt c decreases about seven folds. At the same...
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